Effects of Wickerhamomyces anomalus Co-Fermented with Saccharomyces cerevisiae on Volatile Flavor Profiles during Steamed Bread Making Using Electronic Nose and HS-SPME-GC-MS

Steamed bread is a traditional staple food in China, and it has gradually become loved by people all over the world because of its healthy production methods. With the improvement in people’s living standards, the light flavor of steamed bread fermented by single yeast cannot meet people’s needs. Multi-strain co-fermentation is a feasible way to improve the flavor of steamed bread. Here, the dynamic change profiles of volatile substances in steamed bread co-fermented by Saccharomyces cerevisiae SQJ20 and Wickerhamomyces anomalus GZJ2 were analyzed using the electronic nose (E-nose) and headspace solid-phase microextraction combined with gas chromatography–mass spectrometry (HS-SPME-GC-MS). The five detectors of the E-nose rapidly detected the changes in volatile substances in different dough or steamed bread with the highest response value in co-fermented dough. A total of 236 volatile substances were detected in all the samples using HS-SPME-GC-MS, and alcohols were the most variable component, especially Phenylethyl alcohol. Significantly, more alcohols and esters were upregulated in co-fermented dough, and the addition of W. anomalus GZJ2 improved the key volatile aroma compounds of steamed bread using the relative odor activity value method (ROAV), especially the aldehydes and alcohols. Moreover, these key volatile aroma compounds can be quickly distinguished using the W2S detector of the E-nose, which can be used for the rapid detection of aroma components in steamed bread.


Introduction
Steamed bread is a traditional staple food in China and occupies an important position in the Chinese diet.More than 1.3 billion people in China often eat steamed bread, and it has gradually become loved by people all over the world because of its healthy production methods [1].The starter plays an important role in the process of making steamed bread.Sourdough (also called "Laomian" or "Jiaozi") has been used to make steamed bread for thousands of years in China, which has the advantages of good flavor, excellent texture, and a long shelf-life [2,3], because it contains a lot of yeasts, lactic acid bacteria, and even mold.This also leads to the shortcomings of sourdough steamed bread, such as the complex production process, unstable quality, and difficult industrial large-scale production [4].In recent decades, highly active commercial yeast has been used as the most common starter for making steamed bread, which has the advantages of a simple process and short fermentation time.With the improvement in people's living standards, people began to pay attention to the flavor of staple food.Obviously, the flavor of steamed bread fermented Foods 2024, 13, 2490 3 of 19 however, few studies focus on the effects of other strains on flavor substances, especially aroma components, during steamed bread making.
In our previous work, the mixed fermentation of S. cerevisiae SQJ20 and W. anomalus GZJ2 at a ratio of 1:2, newly isolated from sourdough, significantly improved the quality of steamed bread than those by commercial yeast.Specifically, the addition of W. anomalus GZJ2 increased the content of total protein and free amino acids in steamed bread [9]; however, their volatile flavor substances, especially aroma components, were not studied in detail.Therefore, in this study, the headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) combined E-nose technology was used to analyze the dynamic changes in the volatile flavor compounds during the process of making steamed bread fermented by S. cerevisiae SQJ20 and W. anomalus GZJ2, especially focusing on the effect of W. anomalus GZJ2 on the volatile aroma substances in steamed bread, which will lay a theoretical and application foundation for the industrial production of mixed-starter steamed bread.

Yeast Strains and Culture Conditions
The yeast S. cerevisiae SQJ20 and W. anomalus GZJ2 used in this study were screened and obtained from different Chinese sourdoughs by our laboratory.The yeast strains were cultured on a solid medium at 30 • C for 48 h, then they were inoculated and cultured in a liquid medium for 24 h to obtain the seed liquid.The seed solution was inoculated into a liquid medium at 1% (v/v) and cultured to the later stage of logarithmic growth (S.cerevisiae SQJ20 (12 h) and W. anomalus GZJ2 (24 h)), then the yeast was collected to make the steamed bread.The used liquid medium included yeast extract 10 g/L, peptone 20 g/L, and glucose 20 g/L.

Dough and Steamed Bread Making
Dough making: Wheat flour (Wudeli, Handan China) was mixed evenly with warm water (30 • C) containing the starter at the ratio of 2:1 (w/v) until it was simply clumped together, left standing for about 10 min, and kneaded to a smooth dough, which was used as the control (unfermented dough).Among them, the single starter was S. cerevisiae SQJ20 and the complex starters were S. cerevisiae SQJ20 and W. anomalus GZJ2 at a ratio of 1:2.After fermentation for 90 min at 37 • C, the fermented dough containing S. cerevisiae SQJ20 was abbreviated as SQJ20 (dough), and the fermented dough containing the complex starter S. cerevisiae SQJ20 and W. anomalus GZJ2 was abbreviated as SQJ20 + GZJ2 (dough).
Steamed bread making: The fermented dough was divided into a small dough (75 g) and rounded into the shape of steamed bread.After the secondary fermentation at 30 • C for 40 min, the fermented dough was steamed for 30 min and braised for 10 min, and the steamed bread was obtained.The steamed bread fermented by S. cerevisiae SQJ20 was abbreviated as SQJ20 (steamed bread), and the co-fermented steamed bread (S. cerevisiae SQJ20 and W. anomalus GZJ2) was abbreviated as SQJ20 + GZJ2 (steamed bread).

E-Nose Analysis
The E-nose (PEN3, AirSense, Germany) was used to detect the volatile substances of steamed bread samples, referring to Saa's method [18] with a slight modification.The corresponding sensing substances of each detector in the E-nose are shown in Table 1.The steamed bread sample was divided into 5 mm × 5 mm pieces, then a 3.0 g sample was placed in a sealed glass bottle (30 mL), balanced at 25 • C for 15 min, and tested 10 times on the E-nose.The obtained data were analyzed using a principal component analysis.

HS-SPME-GC-MS
HS-SPME was selected for the pre-treatment of steamed bread before the GC-MS experiments, which is a common extraction method for the volatile flavor analysis [19,20].Specifically, the sample was shaken at a speed of 450 rpm (5 s on, 2 s off) at 60 • C for 15 min, and then the 50/30 µm DVB/CAR on the PDMS extraction head was inserted into the headspace part of the sample, which was maintained in the headspace for 45 min and resolved at 250 • C for 5 min before separation and identification using GC-MS.Before extraction, the extraction head should be aged in the fiber conditioning station for 2 h, and the fiber conditioning station was heated and desorbed for 10 min before and after the sampling.
GC-MS was used to determine the volatile substances in different dough and steamed bread samples, referring to Fan's research method [12].The DB-WAX capillary column (30 m × 0.25 mm × 0.25 µm, Agilent J&W Scientific, Folsom, CA, USA) was selected for the sample injection.The carrier gas was high-purity helium (purity not less than 99.999%) and the constant flow rate was 1.0 mL/min.The inlet temperature was 230 • C, the injection was not divided, and the solvent delay was 1.5 min.The heating procedure was: kept at 40 • C for 1 min, increased to 70 • C at 10 • C/min and held for 2 min, increased to 105 • C at 3 • C/min and held for 2 min, and increased to 180 • C at 10 • C/min and held for 2 min.Finally, the temperature rose to 220 • C at 5 • C/min and it was held for 5 min.The conditions for mass spectrometry were as follows: electron impact ion source (EI), ion source temperature 230 • C, four-stage rod temperature 150 • C, and electron energy 70 eV.The scanning mode was the full scanning mode (SCAN), and the quality scanning range was 20-650 m/z.

ROAV
The relative odor activity value (ROAV) of the volatile compound was calculated using the relative content of the volatile compounds, referring to the method of Wang et al. [21].The ROAV value of the volatile flavor compound with the most significant effect on the overall flavor was defined as 100, and the ROAV values of the other volatile flavor compounds ranged from 0 to 100.Compounds with ROAV ≥ 1 contributed more to the aroma of the sample and were considered as the key volatile favor compounds of the sample.The ROAV was calculated as follows: ROAV = 100 × (C i /C max ) × (T max / Ti ), where C i was the relative content of the volatile compounds, C max was the relative content of the compound with the most obvious influence on the overall flavor, T max was the odor threshold of the compounds with the most obvious influence on the overall flavor, and T i was the odor threshold of the volatile compounds in water, referring to the website of Odor Detection Thresholds & References (leffingwell.com).

Statistical Analysis
A non-targeted metabolomics analysis was performed on the data obtained using GC-MS.The stability of the sample pre-processing, sample loading, and mass spectrometry system was tested using an internal standard and QC sample quality control.The data processing was carried out based on the DIA method of independent data acquisition, and the imported data were processed by peak detection, peak identification, MS2Dec deconvolution, qualitative, peak alignment, normalization, filtering, missing value interpolation, and so on.The NIST database (https://webbook.nist.gov/chemistry/)was used for the qualitative material analysis.The multivariate statistical analysis was performed using the principal component analysis (PCA), the partial least squares discriminant analysis (PLS-DA), and the orthogonal partial least squares-discriminant analysis (OPLS-DA), and the univariate statistical analysis was performed by Student's t test (T-test) and Fold change (FC).According to the VIP > 1 of the first principal component of the OPLS-DA model and the p-value < 0.05 of the T-test, the differential metabolites were screened, which were further used for the cluster analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway enrichment analysis.

Dynamic Changes of Volatile Compounds during the Making of Steamed Bread Using the E-Nose
Based on the principle of bionics, the E-nose can rapidly detect and comprehensively analyze the volatile substances in samples.The principal component analysis (PCA) method was selected to convert and reduce the dimensionality of the response values of the E-nose detectors, so as to analyze the differences among the different samples [22].Figure 1A shows the PCA results of the response values of the E-nose detectors to different samples, including unfermented dough (Control), fermented dough by S. cerevisiae SQJ20 with or without W. anomalus GZJ2, and steamed bread by S. cerevisiae SQJ20 with or without W. anomalus GZJ2.The cumulative variance contribution rate of the principal components PC1 and PC2 reached 98.9% and covered most of the original data, which can be used to compare the volatile substances of different samples.The samples in each group were clustered together, indicating similar volatile components and a good repeatability, and the five groups were well separated, indicating different volatile characteristics of different groups.
Further, the radar map was drawn based on the response values of the 10 detectors of the E-nose to different samples.As shown in Figure 1B, the response values of the control to the 10 detectors were all very low, indicating that the content of the volatile substances in dough before fermentation was very low and may not be enough to be recognized by the E-nose.After fermentation with S. cerevisiae SQJ20 with or without W. anomalus GZJ2, the fermented dough had different response values to multiple similar detectors, including W1W (inorganic sulfide, terpene compounds), W2S (alcohols, aldehydes, and some aromatic compounds), W2W (aromatic compounds, organic compounds of sulfur), W1S (short chain alkane), W5S (nitrogen compounds), and W2W (organic sulfides, aromatic compounds) (Table 1).It indicated that the single or mixed yeast strains produced similar volatile substances with different levels.Notably, the largest increase was seen in the cofermented dough, indicating that the addition of W. anomalus GZJ2 favored the production of more volatile substances.However, when the fermented dough was steamed into steamed bread, the volatile substances were lower than those in dough, which may be due to the decomposition or transformation of the thermal unstable substances caused by the steaming process.Nevertheless, the volatile substances in the co-fermented steamed breads were still higher than those in the steamed breads fermented by S. cerevisiae SQJ20, which is consistent with the results of the sensory evaluation [9].It can be seen that the volatile flavor substances in different samples can be quickly detected by the E-nose, and the aroma can be quickly judged according to the aroma components corresponding to the different detectors.quickly detected by the E-nose, and the aroma can be quickly judged according to the aroma components corresponding to the different detectors.

Dynamic Changes of Volatile Compounds during the Making of Steamed Bread Using HS-SPME-GC-MS
The volatile compounds of the different dough and steamed bread were detected using HS-SPME-GC-MS.The volatile compounds with scores > 70 were qualitatively analyzed using the NIST database, and the results are shown in Figure 2A.From five samples of dough or steamed bread fermented by S. cerevisiae SQJ20 with or without W. anomalus GZJ2, a total of 236 substances in eight categories were detected, including alcohols, aldehydes, ketones, esters, acids, alkanes, heterocycles, and others.Of all the substances, hydrocarbons, alcohols, and esters accounted for 28.8%, 15.3%, and 15.3%, respectively, accounting for 59.4% of the total.The relative abundance stack diagram was drawn according to the relative contents of the various substances, and the dynamic changes in the volatile substances in the process of making steamed bread were observed through the changes in the relative contents of the volatile substances (Figure 2B).Significantly, compared with the control, alcohols had the greatest increase in the fer-

Dynamic Changes of Volatile Compounds during the Making of Steamed Bread Using HS-SPME-GC-MS
The volatile compounds of the different dough and steamed bread were detected using HS-SPME-GC-MS.The volatile compounds with scores > 70 were qualitatively analyzed using the NIST database, and the results are shown in Figure 2A.From five samples of dough or steamed bread fermented by S. cerevisiae SQJ20 with or without W. anomalus GZJ2, a total of 236 substances in eight categories were detected, including alcohols, aldehydes, ketones, esters, acids, alkanes, heterocycles, and others.Of all the substances, hydrocarbons, alcohols, and esters accounted for 28.8%, 15.3%, and 15.3%, respectively, accounting for 59.4% of the total.The relative abundance stack diagram was drawn according to the relative contents of the various substances, and the dynamic changes in the volatile substances in the process of making steamed bread were observed through the changes in the relative contents of the volatile substances (Figure 2B).Significantly, compared with the control, alcohols had the greatest increase in the fermented dough and steamed bread.Moreover, the content of alcohols in the mixed-fermented dough (56%) or steamed bread (53%) was higher than the content in the single-fermented dough (45%) or steamed bread (51%), respectively, which may be caused by the degradation, decarboxylation, and deamination of amino acids in flour by different yeast strains.Among them, the content of Phenylethyl alcohol changed most significantly after fermentation or steaming.In the control, the content of Phenylethyl alcohol was only 0.59%; however, it reached 31.51% in fermented dough by S. cerevisiae SQJ20, and it reached 35.29% in co-fermented dough by S. cerevisiae SQJ20 with W. anomalus GZJ2.After steaming, the content of Phenylethyl alcohol increased to 39.12% and 46.01% in steamed bread fermented by S. cerevisiae SQJ20 alone and with W. anomalus GZJ2, respectively.Phenylethyl alcohol has been proven to have a sweet fragrance of roses and honey, which contributes a lot to the flavor of steamed bread [23,24].In this study, both the fermentation and steaming processes were conducive to the increase in the Phenylethyl alcohol content, and the addition of W. anomalus GZJ2 further promoted the increase in the Phenylethyl alcohol content, which enhanced the flavor of steamed bread.
mented dough and steamed bread.Moreover, the content of alcohols in the mixed-fermented dough (56%) or steamed bread (53%) was higher than the content in the single-fermented dough (45%) or steamed bread (51%), respectively, which may be caused by the degradation, decarboxylation, and deamination of amino acids in flour by different yeast strains.Among them, the content of Phenylethyl alcohol changed most significantly after fermentation or steaming.In the control, the content of Phenylethyl alcohol was only 0.59%; however, it reached 31.51% in fermented dough by S. cerevisiae SQJ20, and it reached 35.29% in co-fermented dough by S. cerevisiae SQJ20 with W. anomalus GZJ2.After steaming, the content of Phenylethyl alcohol increased to 39.12% and 46.01% in steamed bread fermented by S. cerevisiae SQJ20 alone and with W. anomalus GZJ2, respectively.Phenylethyl alcohol has been proven to have a sweet fragrance of roses and honey, which contributes a lot to the flavor of steamed bread [23,24].In this study, both the fermentation and steaming processes were conducive to the increase in the Phenylethyl alcohol content, and the addition of W. anomalus GZJ2 further promoted the increase in the Phenylethyl alcohol content, which enhanced the flavor of steamed bread.In addition, the content of the hydrocarbons also increased significantly after steaming, possibly because the heating promoted the oxidation and degradation of fatty acids [25].Although the relative content of the hydrocarbons in the steamed bread fermented by S. cerevisiae SQJ20 (18%) was higher than that in the co-fermented steamed bread (14%), most hydrocarbons had a higher aroma threshold and thus contributed little to the overall flavor of steamed bread [26].It is worth noting that the relative content of the acids in dough and steamed bread by mixed fermentation was higher than in those fermented by S. cerevisiae SQJ20.Acid substances not only provide some specific flavors, but also activate people's taste receptors, thus affecting people's perception of flavor substances and obtaining a diversified flavor [27].The volatile substances in fermented dough by S. cerevisiae SQJ20 were compared with the control (unfermented dough) using the multivariate statistical analysis OPLS-DA method and the univariate statistical analysis.As shown in Figure 3A, the samples in the same group were relatively concentrated on the t1 axis, but the distance between the different groups was far, indicating that the volatile substances in the two groups were obviously different.Figure 3B is the visual volcanic diagram of univariate statistical analysis.Compared with the control, 24 volatile substances in the fermented dough by S. cerevisiae SQJ20 were significantly upregulated (red dots) and nine volatile substances were significantly downregulated (blue dots) (p < 0.05), indicating that S. cerevisiae SQJ20 produced more volatile metabolites during dough fermentation.
In addition, the content of the hydrocarbons also increased significantly after steaming, possibly because the heating promoted the oxidation and degradation of fatty acids [25].Although the relative content of the hydrocarbons in the steamed bread fermented by S. cerevisiae SQJ20 (18%) was higher than that in the co-fermented steamed bread (14%), most hydrocarbons had a higher aroma threshold and thus contributed little to the overall flavor of steamed bread [26].It is worth noting that the relative content of the acids in dough and steamed bread by mixed fermentation was higher than in those fermented by S. cerevisiae SQJ20.Acid substances not only provide some specific flavors, but also activate people's taste receptors, thus affecting people's perception of flavor substances and obtaining a diversified flavor [27].The volatile substances in fermented dough by S. cerevisiae SQJ20 were compared with the control (unfermented dough) using the multivariate statistical analysis OPLS-DA method and the univariate statistical analysis.As shown in Figure 3A, the samples in the same group were relatively concentrated on the t1 axis, but the distance between the different groups was far, indicating that the volatile substances in the two groups were obviously different.Figure 3B is the visual volcanic diagram of univariate statistical analysis.Compared with the control, 24 volatile substances in the fermented dough by S. cerevisiae SQJ20 were significantly upregulated (red dots) and nine volatile substances were significantly downregulated (blue dots) (p < 0.05), indicating that S. cerevisiae SQJ20 produced more volatile metabolites during dough fermentation.The differential volatile metabolites in the co-fermented dough by S. cerevisiae SQJ20 with W. anomalus GZJ2 were compared with the control.The two groups showed significant differentiation on the t1 axis of the OPLS-DA score chart (Figure 4A), indicating that different volatile substances were produced in the co-fermentation process.As shown in the visual volcano diagram (Figure 4B), 30 volatile compounds were significantly upregulated and 29 were significantly downregulated in the mixed fermentation (p < 0.05), suggesting that the mixed fermentation of S. cerevisiae SQJ20 and W. anomalus GZJ2 produced more volatile substances than those produced by single S. cerevisiae SQJ20.

Effect of Different Yeast Strains on the Formation of Volatile Compounds in the
Combined with the VIP > 1 of the OPLS-DA first principal component and the p < 0.05 of the univariate statistical analysis, significantly different volatile metabolites were screened and clustered into a heat map (Figure 3C).Compared with the control, a total of 31 significantly different volatile substances were screened in the fermented dough by S. cerevisiae SQJ20, of which 23 were upregulated and eight were downregulated.The upregulated 23 volatile compounds included seven alcohols, six acids, three heterocycles, three esters, two aldehydes, one ketone, and one alkane, which were mainly produced by S. cerevisiae SQJ20 during dough fermentation.

Mixed Fermentation by S. cerevisiae SQJ20 and W. anomalus GZJ2
The differential volatile metabolites in the co-fermented dough by S. cerevisiae SQJ20 with W. anomalus GZJ2 were compared with the control.The two groups showed significant differentiation on the t1 axis of the OPLS-DA score chart (Figure 4A), indicating that different volatile substances were produced in the co-fermentation process.As shown in the visual volcano diagram (Figure 4B), 30 volatile compounds were significantly upregulated and 29 were significantly downregulated in the mixed fermentation (p < 0.05), suggesting that the mixed fermentation of S. cerevisiae SQJ20 and W. anomalus GZJ2 produced more volatile substances than those produced by single S. cerevisiae SQJ20.Combined with VIP > 1 of the first principal component of the OPLS-DA and the univariate statistical analysis p < 0.05, the differential volatile metabolites between the co-fermented dough and the control (unfermented) were screened for the cluster analysis.As shown in the heat map (Figure 4C), a total of 49 differential volatile metabolites were screened in the co-fermented dough compared with the control, including 29 upregulated volatile metabolites and 20 downregulated volatile metabolites.The upregulated volatile substances included nine alcohols, seven esters, five acids, three aldehydes, two heterocycles, two alkanes, and one ketone, which were mainly produced by S. cerevisiae SQJ20 and W. anomalus GZJ2 during dough fermentation with more differences in the metabolite changes than those by S. cerevisiae SQJ20 alone.

Effects of W. anomalus GZJ2 on the Formation of Volatile Compounds
To analyze the effects of W. anomalus GZJ2 on the formation of volatile compounds during fermentation, Table 2 shows the upregulated volatile metabolites in the fermented dough by S. cerevisiae SQJ20 alone or with W. anomalus GZJ2 compared with the control.Among the volatile metabolites with FC > 30, the increase in two alcohols and two esters was higher in the co-fermented dough, including Phenylethyl alcohol (90.40), 3-(methylthio)-1-Propanol (34.28),Hexadecanoic acid ethyl ester (83.09), and Linoleic acid ethyl ester (58.16).However, only Phenylethyl alcohol (75.69) and Phenylacetaldehyde (43.59) were found in the fermented dough by S. cerevisiae SQJ20.Among them, Phenylethyl alcohol was the volatile metabolite with the largest difference in both fermented dough by S. cerevisiae SQJ20 alone or with W. anomalus GZJ2, with the highest difference ratio in the mixed fermentation.Phenylethyl alcohol, with rose aroma and honey sweetness, was a typical product in the Ehrlich metabolic pathway of yeast, which was converted from phenylalanine in flour to Phenylacetaldehyde and then to Phenylethyl alcohol.In this study, the upregulation ratio of Phenylacetaldehyde in the fermented dough by S. cerevisiae SQJ20 (43.59) was higher than that in the co-fermented dough (9.10), which was likely because W. anomalus GZJ2 promoted the conversion of Phenylacetaldehyde to more Phenylethyl alcohol during the mixed fermentation process, thus improving the flavor of the co-fermented steamed bread.In addition, Hexadecanoic acid ethyl ester, with cream aroma, was upregulated by 83.09 FCs in the mixed fermented dough, but by only 29.93 FCs in the fermented dough by S. cerevisiae SQJ20 alone.3-(methyl thioyl)-1-Propanol, with the aroma of baked potatoes, was upregulated by 34.28 FCs in the co-fermented dough, but only by 14.85 FCs in the fermented dough by S. cerevisiae SQJ20 alone.Note: FC means fold change.SQJ20 stands for S. cerevisiae SQJ20; SQJ20 + GZJ2 stands for S. cerevisiae SQJ20 and W. anomalus GZJ2.

Key Metabolic Pathways during Dough Fermentation
The enrichment of the KEGG pathway of the differential volatile metabolites, obtained from the fermented dough by S. cerevisiae SQJ20 alone or with W. anomalus GZJ2, was helpful to understand the metabolic pathway changes during fermentation.The same metabolic pathways, including the protein digestion and absorption pathway, and Phenylalanine metabolism pathway, were enriched in the single and mixed yeast fermented dough.Among them, the Phenylalanine metabolic pathway was the main pathway of flavor formation during dough fermentation (Figure 5).Phenylethyl alcohol and Phenylacetaldehyde represented by red dots were the main upregulated metabolites in this metabolic pathway, which was consistent with the results of differential metabolite analysis.

Changes in the Volatile Compounds of Co-Fermented Steamed Bread during the Steaming Process
To analyze the effect of the steaming process on the changes in the volatile components, the volatile substances in steamed bread co-fermented by S. cerevisiae SQJ20 with W. anomalus GZJ2 were compared with those in the co-fermented dough (Figure 6).As shown in the score chart of the OPLS-DA (Figure 6A), the two groups have obvious discrimination on the t1 axis, indicating that the steaming had a great influence on the volatile substances.Figure 6B shows the visual volcano map of the univariate statistical analysis.Compared with co-fermented dough, 20 volatile metabolites were significantly upregulated (red dots) and 26 were significantly downregulated (blue dots) (p < 0.05) in co-fermented steamed bread, indicating that the steaming process significantly affected the content of the volatile metabolites.Compared with co-fermented dough, the differential volatile metabolites in cofermented steamed bread were screened for the cluster analysis.As shown in Figure 6C and Table 3, 35 differential volatile metabolites were screened in steamed bread, including 16 upregulated and 19 downregulated differential metabolites.Among them, the upregulated volatile substances included six alkanes, three esters, two heterocycles, one ketone, one aldehyde, and three other substances, and the downregulated volatile substances included seven esters, five alcohols, two aldehydes, two heterocycles, one acid, one ketone, and one alkane, with more downregulated volatile substances.Due to the instability of heat, some metabolites were transformed or decomposed to form other volatile compounds, in particular, alcohols and esters were more affected by heat.However, they did not form the characteristic flavor compounds such as pyrazines and pyrrole in bread, because the temperature of steaming did not reach the temperature of the Maillard reaction and the caramelization reaction.

Changes in the Volatile Compounds of Co-Fermented Steamed Bread during the Steaming Process
To analyze the effect of the steaming process on the changes in the volatile components, the volatile substances in steamed bread co-fermented by S. cerevisiae SQJ20 with W. anomalus GZJ2 were compared with those in the co-fermented dough (Figure 6).As shown in the score chart of the OPLS-DA (Figure 6A), the two groups have obvious discrimination on the t1 axis, indicating that the steaming had a great influence on the volatile substances.Figure 6B shows the visual volcano map of the univariate statistical analysis.Compared with co-fermented dough, 20 volatile metabolites were significantly upregulated (red dots) and 26 were significantly downregulated (blue dots) (p < 0.05) in co-fermented steamed bread, indicating that the steaming process significantly affected the content of the volatile metabolites.Compared with co-fermented dough, the differential volatile metabolites in co-fermented steamed bread were screened for the cluster analysis.As shown in Figure 6C and Table 3, 35 differential volatile metabolites were screened in steamed bread, including 16 upregulated and 19 downregulated differential metabolites.Among them, the upregulated volatile substances included six alkanes, three esters, two heterocycles, one ketone, one aldehyde, and three other substances, and the downregulated volatile substances included seven esters, five alcohols, two alde- Previous studies have confirmed the transformation of substances during heating.Pico et al. [28] proposed that alcohols, such as 2-methyl-1-propanol and 3-methyl-1-butanol, were largely lost during bread baking, mainly due to the low boiling point of alcohols.Brich et al. [29] also proposed that esters in bread were extremely volatile during baking.In this study, the content of five alcohols ((Z)-3-Nonen-1-Ol, 1-Octanol, 2-Ethyl-1-Hexanol, 1-Nonanol, and 1-Heptanol) in co-fermented steamed bread significantly decreased after steaming, indicating that alcohols were volatile in the heating process.Meanwhile, the contents of seven esters decreased and only three esters showed an increase after steaming, which showed that esters were greatly affected by heating.In the increased esters, the contents of 3-methyl-1-butanol acetate and acetic acid 2-phenylethyl ester increased after steaming, likely due to their higher boiling points (142 • C and 232 • C, respectively) than the steaming temperature (100 • C).Moreover, 3-methyl-1-butanol acetate, with a fruit flavor, and acetic acid 2-phenylethyl ester, with a flower flavor, enhanced the flavor of steamed bread.In addition, the upregulated substances also contained six kinds of alkanes and two kinds of heterocycles, which may be caused by the heat treatment to promote the oxidation of fats.It can be seen that the heating process affects many complex changes, all of which affect the flavor of steamed bread, and the potential complex chemical reactions need to be further studied.

Effect of S. cerevisiae and W. anomalus on the Formation of Volatile Aroma Compounds in Steamed Bread
The key volatile flavor compounds in steamed breads were identified using the relative odor activity value method (ROAV) based on the relative content of the volatile compounds (Table 4), which can evaluate the influence of a single volatile compound on the overall aroma [30][31][32].A total of 12 characteristic aromatic compounds (ROAV > 1) were screened in steamed bread fermented by S. cerevisiae SQJ20 with or without W. anomalus GZJ2, including seven aldehydes, three alcohols, one ester, and one furan.Remarkably, aldehydes made important contributions to the aroma of steamed bread, including Hexanal, Heptanal, Nonanal, Decanal, (E,E)-2,4-Decadienal, (E,E)-2,4-Nonadienal, and Phenylacetaldehyde.Among them, (E,E)-2,4-Decadienal was the volatile aroma substance with the largest ROAV value (ROAV = 100) in steamed bread fermented by S. cerevisiae SQJ20 with or without W. anomalus GZJ2, contributing to the aroma of fried, waxy, and fat, which was related to the lipid oxidation reaction of flour [2,24].Compared with those of steamed bread fermented by S. cerevisiae SQJ20, the ROAVs of (E,E)-2,4-Nonadienal (ROAV = 22.85) and Nonanal (ROAV = 12.16) in the co-fermented steamed bread increased, indicating that the addition of W. anomalus GZJ2 improved the aroma of fat, citrus, green, and wax.Meanwhile, the ROAV of Decanal in co-fermented steamed bread decreased compared with that of steamed bread fermented by S. cerevisiae SQJ20, and the ROAVs of Hexanal and Heptanal had similar values, which were the common flavor substances in steamed bread that come from the flour itself [26].A further correlation analysis between aroma substances and aroma characteristics in the co-fermented steamed bread (Figure 7) confirmed that fat aroma was prominent and associated with five aldehydes, indicating that aldehydes were important sources in complex fermented steamed bread.Obviously, aldehydes and alcohols accounted for most of the volatile aroma components in co-fermented steamed bread, indicating that the main aroma substances of co-fermented steamed bread were aldehydes and alcohols, which were also the main aroma substances of the single-fermented steamed bread.Combined with the rapid detection results of the E-nose, it was found that, among the five detectors with response values for the volatile substances in co-fermented steamed bread, W2S mainly responded to aroma substances such as alcohols, aldehydes, and ketones, indicating that W2S was the main response detector for sensing steamed bread aroma components.This shows that the E-nose can be used to quickly distinguish whether the steamed bread aroma is rich, which can mainly refer to the response value of the W2S.Although the GC-MS Alcohols also occupied an important position in the aroma of steamed bread, including Phenylethyl alcohol, 1-Octen-3-ol, and 1-Heptanol.1-Octen-3-Ol, with mushroom fragrance, and had higher ROAVs in both kinds of steamed bread (49.31, 50.07), indicating that 1-Octen-3-Ol was an important volatile aroma component in steamed bread.It is worth noting that the ROAV of Phenylethyl alcohol (ROAV = 4.84) in co-fermented steamed bread was higher than that of steamed bread fermented by S. cerevisiae SQJ20 (ROAV = 3.58).In fact, the relative content of Phenylethyl alcohol in steamed bread fermented by S. cerevisiae SQJ20 with or without W. anomalus GZJ2 was relatively higher, which was 39.12% and 46.01%, respectively.The smaller ROAV of Phenylethyl alcohol was due to its higher threshold (750 µg/L).Phenylethyl alcohol has mixed aroma characteristics of honey, spice, rose, and lilac, and it is a typical alcohol produced by the yeast Ehrlich pathway, which is derived from phenylalanine in flour and converted into Phenylacetaldehyde and finally into Phenylethyl alcohol [24,33,34].After the addition of W. anomalus GZJ2, the ROAV of Phenylethyl alcohol in steamed bread increased, while the ROAV of Phenylacetaldehyde decreased, indicating that W. anomalus GZJ2 strengthened the metabolism of the Ehrlich pathway and helped convert Phenylacetaldehyde into Phenylethyl alcohol.
Obviously, aldehydes and alcohols accounted for most of the volatile aroma components in co-fermented steamed bread, indicating that the main aroma substances of co-fermented steamed bread were aldehydes and alcohols, which were also the main aroma substances of the single-fermented steamed bread.Combined with the rapid detection results of the E-nose, it was found that, among the five detectors with response values for the volatile substances in co-fermented steamed bread, W2S mainly responded to aroma substances such as alcohols, aldehydes, and ketones, indicating that W2S was the main response detector for sensing steamed bread aroma components.This shows that the E-nose can be used to quickly distinguish whether the steamed bread aroma is rich, which can mainly refer to the response value of the W2S.Although the GC-MS analysis has the advantage of a comprehensive analysis of volatile substances, it has the disadvantages of a high cost, slow analysis, and it cannot directly display aroma components.In contrast, the advantages of the E-nose are also prominent: low cost, rapid detection, and it comprehensively determine the aroma category.Of course, it is necessary to conduct a correlation analysis of the data of the E-nose and the GC-MS in order to use the E-nose to quickly judge the aroma more accurately.Many other studies have also confirmed the advantages of E-noses [35,36].
In addition, the ROAV of 2-Pentyl-furan, with green bean and butter fragrance, was significantly higher in co-fermented steamed bread (ROAV = 6.99) than that in SQJ20 fermented steamed bread (ROAV = 0.33), indicating that W. anomalus GZJ2 contributed to the formation of 2-Pentyl-furan.Moreover, the addition of W. anomalus GZJ2 significantly increased the content of some other volatile flavor compounds in co-fermented steamed bread, which were not represented in Table 4 due to the absence of threshold values, such as 3-(methylthio)-1-Propanol (baked potato), 2-Methyl-butanoic acid (sweet, cheese), and 3-Hydroxy-butanoic acid ethyl ester (fruity) [37].Previously, Liu et al. [34] showed that steamed bread made by W. anomalus possessed a high quantity of 3-(methylthio)-1propanol, which implied an active metabolism via the Ehrlich pathway in W. anomalus.In the metabolism of the Ehrlich pathway, methionine in flour can be converted to the corresponding aldehydes and alcohols [38].Yan et al. [39] confirmed that steamed breads fermented by type I sourdough had a higher relative concentration of specific volatile compounds, including acids, furans, aldehydes, and their corresponding alcohols, such as Acetic acid, 2-Pentyl furan, Hexanal, Nonanal, 2-Octenal, and 2-Nonenal.Overall, the addition of W. anomalus GZJ2 improved the quality of steamed bread, which was reflected in its increased volatile aroma compounds, total protein and free amino acid content, reduced pH and TTA, and softer and more palatable texture, which were confirmed by the electronic nose and sensory evaluation [9].It can be seen that the addition of W. anomalus GZJ2 improves the quality of steamed bread on the whole.These studies provide an important theoretical and practical basis for the application of W. anomalus GZJ2 in the fermentation of flour products.

Conclusions
In this study, the dynamic changes in the volatile components during steamed bread making were detected using the E-nose and HS-SPME-GC-MS, and the effects of Wick-erhamomyces anomalus co-fermented with Saccharomyces cerevisiae on the volatile flavor profiles in dough or steamed bread were analyzed.The E-nose can rapidly detect and comprehensively analyze the volatile substances in different dough or steamed bread in this study or others [40] with different response values, and the response value of co-fermented dough or steamed bread was higher than that of S. cerevisiae SQJ20 alone.Based on the HS-SPME-GC-MS non-targeted metabolomics analysis, 236 volatile compounds were detected in all samples.The dynamic changes in the volatile substances were analyzed during steamed bread making, with alcohols being the largest component, especially Phenylethyl alcohol.The effects of different yeasts on the formation of volatile components in dough were further compared.Compared with fermented dough by S. cerevisiae SQJ20, more alcohols and esters substances were upregulated in co-fermented dough by W. anomalus GZJ2 and S. cerevisiae SQJ20, with the Phenylalanine metabolic pathway as the key KEGG pathway.The steaming process also caused significant changes in the volatile substances in co-fermented steamed bread.Finally, the key volatile flavor compounds in steamed breads were identified using the relative odor activity value method (ROAV).The addition of W. anomalus GZJ2 improved the volatile flavor compounds of steamed bread, especially the aldehydes and alcohols, which can be quickly distinguished using the W2S detector of the E-nose.Moreover, the addition of W. anomalus GZJ2 increased the content of total protein and free amino acids in steamed bread [9].Therefore, the co-fermentation of W. anomalus GZJ2 and S. cerevisiae SQJ20 is expected to be used in the industrial production of flavored steamed bread.

Figure 1 .
Figure 1.E-nose analysis of different dough and steamed bread fermented by S. cerevisiae SQJ20 with or without W. anomalus GZJ2.(A) Principal component diagram; (B) Radar map.

Figure 1 .
Figure 1.E-nose analysis of different dough and steamed bread fermented by S. cerevisiae SQJ20 with or without W. anomalus GZJ2.(A) Principal component diagram; (B) Radar map.

Figure 2 .
Figure 2. Dynamic changes in volatile substances and their contents in the dough or steamed bread fermented by S. cerevisiae SQJ20 with or without W. anomalus GZJ2.(A) The amount of various volatile substances; (B) Stack diagram of the relative abundance of volatile substances.

Figure 2 .
Figure 2. Dynamic changes in volatile substances and their contents in the dough or steamed bread fermented by S. cerevisiae SQJ20 with or without W. anomalus GZJ2.(A) The amount of various volatile substances; (B) Stack diagram of the relative abundance of volatile substances.

Figure 3 .
Figure 3.Effect of S. cerevisiae SQJ20 (In short: SQJ20) on the formation of volatile substances in fermented dough compared with the unfermented dough (Control).(A) OPLS-DA score chart; (B) Volcanic maps; (C) Heat map.

Figure 3 .
Figure 3.Effect of S. cerevisiae SQJ20 (In short: SQJ20) on the formation of volatile substances in fermented dough compared with the unfermented dough (Control).(A) OPLS-DA score chart; (B) Volcanic maps; (C) Heat map.Combined with the VIP > 1 of the OPLS-DA first principal component and the p < 0.05 of the univariate statistical analysis, significantly different volatile metabolites were screened and clustered into a heat map (Figure3C).Compared with the control, a

Foods 2024 ,
13, x FOR PEER REVIEW 13 of 19such as pyrazines and pyrrole in bread, because the temperature of steaming did not reach the temperature of the Maillard reaction and the caramelization reaction.

Figure 6 .Table 3 .
Figure 6.Effect of steaming on the formation of volatile substances in co-fermented steamed bread compared to dough co-fermented by W. anomalus GZJ2 and S. cerevisiae SQJ20.(A) OPLS-DA score chart; (B) Volcanic maps; (C) Heat map.

Foods 2024 , 19 Figure 7 .
Figure 7. Correlation network diagram of sensory flavor characteristics and flavor substances in co-fermented steamed bread.

Figure 7 .
Figure 7. Correlation network diagram of sensory flavor characteristics and flavor substances in co-fermented steamed bread.

Table 1 .
Corresponding sensing substances of each detector of the E-nose.

Table 2 .
Upregulated differential volatile metabolites in fermented dough by S. cerevisiae SQJ20 with or without W. anomalus GZJ2 compared with unfermented dough.